FR2918276A1 - Use of polysaccharide gel from natural origin comprising aqueous composition of polysaccharides and biocompatible alcohols for the preparation of injectable formulation to treat articular degeneration and osteoarthritis - Google Patents

Abstract

Use of polysaccharide gel from natural origin comprising an aqueous composition of polysaccharides (1-100 mg/ml) and biocompatible alcohols (0.0001-100 mg/ml), obtained by the preparation of an aqueous solution of polysaccharides and alcohols by sterilization of the solution, for the preparation of an injectable formulation used in the treatment of articular degeneration, is claimed. ACTIVITY : Antiarthritic; Osteopathic. MECHANISM OF ACTION : None given.

Description

The invention relates to the use of a polysaccharide gel (s)

  natural composition (s) comprising an aqueous composition of one or more polysaccharide (s) and one or more biocompatible alcohol (s) for the preparation of an injectable formulation for the treatment of joint degeneration.

  A joint is a junction connecting two bones and giving them mobility relative to each other. The synovial joints are the most numerous joints, especially in the limbs. In these joints, the bones unite via a cavity filled with a viscous and elastic liquid called synovial fluid. Synovial fluid is responsible for the proper functioning and protection of the joints. It consists in particular of a polysaccharide, hyaluronic acid, which gives the synovial fluid viscoelasticity properties allowing, according to the imposed constraints, a lubrication of the joint or a shock absorption. In the case of joint degenerations such as osteoarthritis of the knee (degeneration due in particular to factors such as obesity, heredity, trauma, ...), the synovial fluid is degraded (decrease in concentration and mass Hyaluronic acid molecule) and this degradation reduces the ability of the synovial fluid to lubricate the joint and to cushion shocks. Viscosupplementation treatment consists of injecting a gel into the joint in order to replace the deficient synovial fluid. Viscosupplementation can reduce or eliminate pain and help restore joint mobility. Viscosupplementation products currently on the market are gels that contain hyaluronic acid. These gels can be based on hyaluronic acid of animal or non-animal origin and crosslinked (case of Synvisc, Durolane) or non-crosslinked (case of Synocrom, Arthrum, Lubravisc, Structovial).

  It is well known to those skilled in the art that the persistence of a gel based on hyaluronic acid is low in a joint (from a few hours to a few days). This low remanence is explained in particular by the mechanical and radical deterioration of the gel (osteoarthritic joints are often the seat of a strong radical activity). Although the therapeutic efficacy of the viscosupplement is of longer duration than its residence time in the joint, the persistence of a hyaluronic acid-based gel in the joint is a preponderant parameter governing the effectiveness of the product. Thus, the longer the hyaluronic acid-based gel has a significant residence time in the joint and the more viscosupplementation treatment (pain reduction, mobility gain) is effective. The current viscosupplementation products used in the treatment of joint degenerations very rarely have a rheology perfectly adapted to the mechanical constraints of the joints (see Example 6) and none of these products contain a molecule capable of limiting the radical degradation and thus of to improve the remanence of the gel within the joint (and consequently the therapeutic effectiveness of the viscosupplement). The present invention consists of a viscosupplement based on polysaccharide (s) and alcohol (s) which has, in some cases, a rheology close to that of the synovial fluid and always an increased resistance to degradation (radical and thermal) by compared to a gel based on polysaccharide (s) conventional, that is to say a gel consisting solely of polysaccharide (s) in aqueous solution (= polysaccharide gel (s) without adding one or more alcohol (s) biocompatible (s)). Used in the preparation of an injectable formulation, it has many interests in the treatment of joint degeneration. It is known from French application No. 06 00138 that a natural polysaccharide gel comprising an aqueous solution of polysaccharide and a biocompatible viscous alcohol has, after sterilization, an increased viscosity which improves its rejuvenating action of the skin after injection (indication: dermatology). However, it has unexpectedly been found that for a particular composition of an aqueous solution of one or more uncrosslinked or substantially uncrosslinked natural polysaccharide (s) and one or more alcohol (s) (s) biocompatible (s), sterilization gives this gel viscoelastic properties quite amazing in that they practically reproduce the viscoelastic properties of healthy synovial fluid. In addition, it has been shown that the presence of one or more biocompatible alcohol (s) in a crosslinked or non-crosslinked polysaccharide gel (s) induces, on the one hand, a increased resistance to free radicals and secondly, a better resistance to degradation during aging (storage of the gel) compared to a polysaccharide gel (s) not containing alcohol (s) biocompatible (s).

  These properties (rheology and increased resistance to degradation) give this new formulation a major interest in the treatment of joint degeneration. Therefore, according to one aspect of the present invention, an injectable, ready-to-use gel consisting of an aqueous solution of one or more natural polysaccharide (s) uncrosslinked (s) or substantially uncrosslinked ( s) and one or more biocompatible alcohol (s), has the following major advantages for use in the treatment of joint degeneration: the viscoelastic properties of sterile gel are close to that of healthy synovial fluid. Thus, the gel has a lubricating role of the joint with low shear stress and a protective role of the joint (shock absorption) at high shear frequency. The rheology of this gel is therefore perfectly adapted to the mechanical stresses exerted in a joint. the gel has an increased resistance to radical degradation (compared to a gel based on biocompatible alcohol-free polysaccharide (s)) due to the antioxidant activity of the biocompatible alcohol. This high resistance to degradation by free radicals induces a greater remanence of the gel in the joint and therefore an increased efficiency of the viscosupplement. the gel has a better resistance to degradation during aging (thermal degradation in particular). This increased resistance makes it possible to improve the conservation of the rheological properties of the gel during storage at ambient temperature and thus to increase the shelf life of the product.

  The publication MAllUCCO D. et al., Rheology of joint fluid in total knee arthroplasty patients; Journal of Orthopedic Research, 1157-1163, 2002, indicates that the crossover frequency between the elastic modulus G 'and the viscous modulus G "is equal to 0.41 0.12 Hz for a healthy synovial fluid (non-arthosed) of the knee - Below 0.41 Hz: G "> G ', the gel is mainly viscous involving a strong lubrication of the joint when the patient is at rest. Above 0.41 Hz: G '> G ", the gel is predominantly elastic, implying a strong shock absorption when the patient is running or jumping, According to the present invention, the gel consisting of an aqueous solution of or several natural polysaccharide (s) and one or more biocompatible alcohol (s) has, after sterilization, a cross frequency fo between the elastic modulus G 'and the viscous module G "close to 0, 41 Hz (see example 3). Thus, the gel has viscoelastic properties close to that of the synovial fluid.

  Therefore, according to the present invention: - Below fo: G "> G ', the gel is predominantly viscous with effective lubrication of the joint at rest, - Above fo: G'> G", the gel is predominantly elastic with an effective absorption of shocks when the patient runs or jumps (protection of the joint).

  Such a rheology is therefore appropriate to the mechanical constraints of the joints and in particular the knee, the hip or the small joints. Therefore, it is of great interest in the treatment of osteoarthritis by viscosupplementation of the knee or other joints.

  Figures 1-5 show the viscous and elastic modules of gels according to the present invention while Figures 6-9 show those of commercial gels. According to another aspect of the present invention, the gel consisting of an aqueous solution of one or more crosslinked or non-crosslinked natural polysaccharide (s) and of one or more alcohol (s) biocompatible (s) has a strong resistance to degradation by free radicals. This high resistance to degradation by free radicals is induced by the presence of the biocompatible alcohol, which by its antioxidant property, captures the free radicals and thus protects the polysaccharide gel (s) (see Example 4 / example 7).

  The biocompatible alcohols are preferably chosen from the family of polyols (for example: glycerol, propylene glycol, sorbitol, mannitol or cyclic monosaccharides such as glucose). The gel of the present invention is more resistant to degradation by free radicals. Therefore, in vivo, it retains its rheological properties for a longer time compared to a biocompatible alcohol-free gel. This longer remanence of the gel with respect to free radicals is to be correlated with a better efficiency of the viscosupplement injected into the joint. Furthermore, the gel of the present invention consisting of an aqueous solution of one or more crosslinked or non-crosslinked natural polysaccharide (s) and one or more biocompatible alcohol (s) ( s) has an increased resistance to degradation during aging of the gel (thermal degradation in particular). When storing at room temperature a polysaccharide gel (s), the rheological properties are modified because of the mainly thermal degradation of the gel. The addition of one or more biocompatible alcohol (s) in the gel makes it possible to slow down these rheological modifications by reducing the degradation of the product (see Example 5). The alcohol plays, in this case, a thermostabilizing role of the polysaccharide gel (s). This resistance to degradation (thermal degradation in particular) makes it possible to improve the conservation of the rheological properties of the gel during storage at ambient temperature and thus to increase the shelf life of the product. The invention therefore relates to the use of a natural polysaccharide gel (s) comprising an aqueous solution of one or more polysaccharide (s) at 100 mg / ml and of one or more alcohol (s). ) biocompatible at 0.0001 - 100 mg / ml.

  This gel is obtained first of all by preparing an aqueous solution composed of one or more polysaccharide (s) and one or more biocompatible alcohol (s) and then by sterilization (aseptically or at the same time). autoclave) of this solution, preferably in an autoclave. The injectable formulation thus obtained is used in the treatment of joint degeneration.

  The polysaccharide of natural origin, alone or as a mixture, is chosen from hyaluronic acid, chondroitin sulfate, keratan, keratan sulfate, heparin, heparan sulfate, cellulose and its derivatives, chitosan, xanthanes, alginates, and all of their respective salts. The polysaccharide of natural origin, alone or as a mixture, is preferably hyaluronic acid or one of its salts, in particular sodium hyaluronate. This polysaccharide is preferably obtained by biofermentation but it can also be of animal origin. Its molecular weight is 0.1 to 10x106Da and preferably 2 to 3x106Da. The polysaccharide concentration is between 1 and 100 mg / ml and preferably between 10 and 25 mg / ml in the case where the polysaccharide is hyaluronic acid or one of its salts. The biocompatible alcohol, alone or as a mixture, is preferably chosen from the family of polyols (for example: glycerol, propylene glycol, sorbitol, mannitol or cyclic monosaccharides such as glucose).

  The concentration of biocompatible alcohol is between 0.0001 and 100 mg / ml and preferably between 15 and 45 mg / ml. The aqueous solution used is preferably a buffered solution. The composition of this buffer solution is chosen in order to have the desired physicochemical and rheological properties. Preferentially, the buffer solution chosen is a phosphate buffer solution. The formulation according to the present invention is used by injection into the viscosupplement joint and the injected dose may be between 0.1 and 20 ml depending on the nature of the treated joint.

  When the polysaccharide (s) are non-crosslinked, or substantially non-crosslinked, on the one hand, the rheology of the gel is perfectly adapted to the mechanical stresses exerted in a joint because close to that of the liquid synovial and secondly, the duration of action of the viscosupplement is prolonged because of the anti-radical action of the biocompatible alcohol (inducing an increase in the remanence of the gel in the joint). Finally, the increased resistance to the mainly thermal degradation of the gel allows a better conservation of the rheological properties of the gel during storage at room temperature and thus an increase in the shelf life of the product.

  As an illustration, two formulations of viscoelastic gels that can be prepared according to the present invention are given below: - Viscoelastic gel based on hyaluronic acid and glycerol Sterile solution composed of 20 mg / ml of hyaluronic acid ( MM = 2.5x106 D) and 20 mg / ml glycerol in phosphate buffer. - Viscoelastic gel based on hyaluronic acid and sorbitol Sterile solution consisting of 20 mg / ml of hyaluronic acid (MM = 2.5x106 D) and 40 mg / ml of sorbitol in phosphate buffer.

  Examples: Examples are provided to illustrate the invention but are not limiting of said invention.

  EXAMPLE 1 Preparation of Formulations According to the Present Invention The formulations according to the present invention are prepared by mixing the polysaccharide (s) and the biocompatible alcohol (s) in a phosphate buffer solution by filling the gel. in glass syringes and sterilizing these syringes in moist heat (T = 121 C) Formulation A: mg hyaluronic acid 2.5x106 D mg glycerol - Qsp 1 ml phosphate buffer Formulation B: 20 mg of hyaluronic acid 2.5x106 D 20 mg of glycerol - Qsp 1 ml of phosphate buffer Formulation C: 20 - 20 mg of hyaluronic acid 2.5x106 D mg of propylene glycol - Qsp 1 ml of phosphate buffer Formulation D: mg of hyaluronic acid at 2.5x106 D - 15 mg of mannitol - Qsp 1 ml of phosphate buffer 8 Formulation E: - 20 mg of hyaluronic acid at 2.5x106 D 40 mg of sorbitol Qsp 1 ml of phosphate buffer

  EXAMPLE 2 Physicochemical Properties of the Formulations of Example 1 pH (at room temperature) Formulation pH A 7.0 B 7.2 C 7.1 D 7.0 E 7.1 Osmolarity Formulation Osmolarity (mOsm / kg) A 335 B 322 C 324 D 315 E 326 Formulations A, B, C, D and E are isotonic and have a neutral pH.

  Example 3: Rheological Properties of the Formulations of Example 1 The viscoelastic properties of formulations A, B, C, D and E are characterized by measuring the evolution of the viscous modulus (G ") and of the elastic modulus (G ') in function frequency (see Figures 1 to 5).

  For these 5 formulations, it is found that the crossing frequency of the module G 'and of the module G "is close to that of the healthy synovial fluid The table below gives the crossover frequency values fc for each formulation and for the liquid healthy synovial Formulation Crossover Frequency (Hz) A 0.50 B 0.32 C 0.32 D 0.32 E 0.33 Healthy synovial fluid 0.41 0.12 (published by Mazzucco D. et al.) As described in the present invention: - Below fc: G "> G ', the gel is predominantly viscous with effective lubrication of the joint at rest, - Above fc: G'> G", the gel is predominantly elastic with effective shock absorption when the patient runs or skips Example 4: Resistance to free radical degradation of the formulations of Example 1 To demonstrate that the presence of a biocompatible alcohol in a polysaccharide gel (s) makes it possible to reduce the degradation of a gel by free radicals; resisting the degradation by free radicals of hyaluronic acid / biocompatible alcohol gels (formulations of Example 1) and the resistance to degradation by free radicals of a gel based on hyaluronic acid without addition of biocompatible alcohol (formulation F of Example 5)). The degradation test is carried out by adding an oxidant in the gel to be tested, homogenizing with a spatula for 1 minute and measuring the value of the tanδ = G "/ G 'parameter at 0.7 Hz (parameter characteristic of the viscoelastic properties of the gel ) at different times (t = 0, 15 min and 30 min) It is found that this parameter increases over time, synonymous with a progressive destructuring of the gel The values measured for the formulations B, C, D, E and F are given in the table below Tanb Formulation (t = 0 min) Tanb (t = 15 min) A Tanb (%) B 1.10 3.34 + 204% C 1, 08 3.13 + 205% D 1.19 4.63 + 289% E 1.08 2.57 + 138% F 1.41 6.56 + 365% As described in the present invention, each formulation B, C, D and E has a radical resistance. greater than that of the biocompatible alcohol-free gel (formulation F), therefore biocompatible alcohols protect the gel effectively against free radical degradation. res.

  EXAMPLE 5 Accelerated aging study of a formulation with and without alcohol Two formulations are put into accelerated aging in an oven at 40 ° C: Formulation B of Example 1: solution based on hyaluronic acid and glycerol formulation F without addition of alcohol (prepared under conditions identical to formulation B of Example 1) - 20 mg of 2.5 × 10 6 hyaluronic acid D - Qsp 1 ml of phosphate buffer A measurement of the zero viscosity (viscosity at zero shear) and a determination of the crossing frequency fc between the elastic modulus G 'and the viscous modulus G "is carried out at 3 times (t = 0, 7 days, 26 days). The results obtained are given in the table below. below: Number of days Formulation Viscosity Change in fo (Hz) Change in fc aging zero viscosity compared to 40 C zeropar compared to (%) to (%) B 252 / 0.32 / O day F 192 / 0.39 / Formulation B * ND / 0.32 0% 7 days F * NA / 0.39 0 % Formulation B 210 - 17% 0.37 + 16% 26 days _ F 143 -26% 0.50 + 28% * ND: not determined It is found that during accelerated aging, the loss of zero viscosity and the shift of the crossover frequency fc are less important in the case of the formulation B (formulation according to the present invention) than in the case of the conventional formulation (formulation F)) Example 6: Comparison of the rheology of 4 commercial viscosupplementation products and The following are the products tested: Product Acid concentration Molecular weight of Hyaluronic sterilization method (mg / ml) Hyaluronic acid (Da) P11 2,2 2,2,7,106 Steam sterilization water P22 10 2.2 - 2.7.106 Sterilization with water vapor P3i 10 1.106 Sterilization by filtration P44 10 4.106 N D6 Formulation A 2.5.106 Heat sterilization of Example 1 (+ 20 mg / ml of glycerol) wet 1 P1: Synocrom (fabr icant: CROMA PHARMA) 2 P2: Structovial (manufacturer: CROMA PHARMA) P3: Fermathron (manufacturer: HYALTECH) 4 P4: Lubravisc (manufacturer: BOHUS BIOTECH) 5 ND: not determined For the 5 gels tested, figures 6 to 9 give the viscous (G ") and elastic (G ') modules as a function of frequency. It can be seen that only the gel according to Example 1 has a crossing frequency (0.50 Hz) close to that of the healthy synovial fluid (0.41 Hz). The table below groups together the crossover frequency values ff for the products P1 to P4 and for the formulation A of Example 1. Product fc (hz) Formulation A of Example 1 0.50 P1 5.8 P2 5, 0 P3 6.3 P4 0.09 13 It is known from the publication of Mazzucco D. et al. (cited above) that the crossing frequency of healthy synovial fluid (0.41 Hz) is below the observed frequencies in the knee during walking (0.7 Hz) and stroke (3 Hz).

  For products P1 to P3, the crossover frequency is well above 3 Hz and therefore, the products do not have a high elasticity for shock absorption when the knee is moving. The product P4 has a very low crossing frequency, the elastic modulus is higher than the viscous modulus over the entire frequency range 0.1 to 10 Hz.

  Therefore, elasticity is important when the knee is moving but the lubrication of the joint is inefficient when the patient is at rest. Example 7: Case of a Formulation Based on a Crosslinked Polysaccharide and an Alcohol The formulation is prepared in the following manner: preparation of a gel based on poysaccharide (s) crosslinked with biocompatible alcohol (s). The crosslinking of the biopolymer is carried out according to techniques well known to those skilled in the art (for example, US4973683, EP1303542, EP0939086, EP0839159, US4716154, US5676964, US6703444, US4605691, EP0272300, EP1611160, EPO408731). filling of the gel in glass syringes and sterilization of these syringes in an autoclave (T = 121 C)

  Let FI, a gel based on hyaluronic acid (HA: MM = 2.4 × 10 6 Da, 10 mg / ml) crosslinked at 10/0 (mass / mass) using BDDE 2 g of sorbitol is added in 10 g of gel A. The whole is mixed with a spatula for 10 minutes. Let F2 be the gel thus obtained. The F1 gel and the F2 gel are filled in glass syringes and then sterilized in the same autoclave charge at 121 C.

  A test for resistance to degradation by free radicals is carried out as described in Example 4.

  It is found that the crosslinked hyaluronic acid gel containing sorbitol (gel F2) has a resistance to degradation by free radicals greater than that of crosslinked hyaluronic acid gel containing no sorbitol (F1 gel). .

  Consequently, a gel based on crosslinked or non-crosslinked polysaccharide (s) containing a biocompatible alcohol has a resistance to degradation by free radicals greater than that of a gel based on polysaccharide (s). crosslinked or uncrosslinked (s) not containing biocompatible alcohol.

Claims (9)

  1. Use of a naturally occurring polysaccharide gel (s) comprising an aqueous composition of one or more polysaccharide (s) at 1 to 100 mg / ml and one or more biocompatible alcohol (s) at 0.0001 mg / ml, obtained by preparation of an aqueous solution of one or more polysaccharide (s) and of one or more alcohol (s) and then by sterilization of this solution, for the preparation of an injectable formulation used in the treatment of joint degeneration.   2. Use of a polysaccharide gel (s) according to claim 1, wherein the one or more polysaccharides are uncrosslinked or substantially uncrosslinked.   3. Use according to claim 1 or 2, wherein the concentration of polysaccharide (s) is 10 to 25 mg / ml and the concentration of alcohol (s) is 15 to 45 mg / ml.   4. Use according to any one of claims 1 to 3, wherein the polysaccharide, alone or in mixture, is hyaluronic acid (or one of its salts) having a molecular weight of 0.1 to 10x106Da and the alcohol, alone or as a mixture, is glycerol, propylene glycol, sorbitol, mannitol or glucose.   5. Use according to claim 4, wherein the hyaluronic acid has a molecular mass of 2 to 3x106Da.   6. Use according to either of claims 4 and 5, wherein the concentration of hyaluronic acid is 20 mg / ml and that of glycerol is 20 mg / ml.   The use according to either one of claims 4 and 5, wherein the concentration of hyaluronic acid is 20 mg / ml and the concentration of sorbitol is 40 mg / ml.   8. Use according to any one of claims 1 to 6 wherein the sterilization is performed by autoclaving. 16   9. Use according to any one of claims 1 to 8 wherein the formulation obtained is injected at a rate of 0.1 to 20 ml